It is known that some microorganisms having nitrilase activity have the ability to convert acrylonitrile to acrylamide through hydration. Such microorganisms so far known include microorganisms belonging to the genera Bacillus, Bacteridium, Micrococcus and Brevibacterium (e.g.,see U.S. Pat. No. 4,001,081, British Pat. No. 1,535,307, German OLS (laid open) No. 2,556,701, and French Pat. No. 2,294,999) the genera Corynebacterium and Nocardia (e.g., see U.S. Pat. No. 4,248,968, British Pat. No. 2,018,240, German OLS (laid open) No. 2,921,292, and French Pat. No. 2,421,212) and the genus Pseudomonas (e.g., see European Pat. No. 93.782).
In producing acrylamide from acrylonitrile using such microorganisms, acrylonitrile is subjected to catalyiic reaction in an aqueous medium (e.g. water, physiological saline, phosphate buffer) using the microbial cells either as is or immobilized, for instance, with a polymer gel. For smooth progress of the enzymatic reaction, the following conditions are generally used: a substrate acrylonitrile concentration of from 0.1 to 10 percent by weight; a cell concentration of from 0.01 to 10 percent by weight; a PH of from 7 to 9; a temperature range of from the freezing point of the aqueous medium to 30.degree. C.; and a time period of from 0.5 to 100 hours.
Currently, semibatchwise or continuous processes using granular immobilized cells are preferred from the viewpoints of prevention of impurity migration from the cells, separability of the cells from reaction mixtures, reusability of the cells, increased enzyme stability, etc., and thus are widely used in carrying out microbial reactions. Such processes are also economically advantageous in the microbial production of acrylamide, and the present inventors have already provided a method for producing acrylamide by continuous column reaction using immobilized cells entrapped with a polyacrylamide gel, for instance (e.g., see Japanese Patent Publication No. 1234/82 and U.S. Pat. No. 4,248,968).
However, irrespective of whether the cells are used as is or in the immobilized state, the use of the abovementioned physiological saline, phosphate buffer, or the like as the aqueous medium is not preferable from the viewpoint of quality, since use thereof means that the product, i.e., the aqueous acrylamide solution, will contain sodium chloride, phosphates, etc., in large amounts. In particular, the presence of phosphates may result in unfavorable results, for example in that, in producing acrylamide polymers having a high degree of polymerization, the polymers obtained are often more or less insoluble in water. Therefore, in such a case, some after-treatment for removing the salts, such as an ion exchange treatment, becomes essential. This makes the operational procedure complicated and troublesome, and hence is unfavorable from an economic viewpoint.
On the other hand, the use of water alone as the aqueous medium also presents problems. For instance, when the reaction is carried out in a water system without using physiological saline or phosphate buffer, the enzyme activity of the cells is apt to fall rapidly. Moreover, when the hydration reaction is carried out continuously using a column packed, for instance, with polyacrylamide gel-immobilized cells, the immobilized cells in said column becomes swollen in a short period of time after the start of the reaction, whereby smooth operation of the procsss becomes impossible.
Thus, in the microbial production of acrylamide, contradictory problems are encountered. When much importance is attached to economic efficiency, and hence stabilization of the cellular enzymes is aimed at, a large amount of salts must be present in the reaction mixture; accordingly, the purity of the product, i.e., the aqueous acrylamide solution, is decreased. When, on the other hand, the purity (quality) of the aqueous acrylamide solution is regarded as more important, the stability of the cellular enzymes tend to be decreased under the conditions that have been used, so that the cost of cell preparation accounts for a great part of the cost of acrylamide production, which is economically disadvantageous.
To overcome such problems, the present inventors previously conducted investigations to try to find a method which might allow the desired reactions to proceed under those conditions employable in producing high-purity aqueous acrylamide solutions, while simultaneously retaining the stability of cellular enzymes for a long period of time. As a result, there was proposed (1) a method which comprises carrying out the reaction in an aqueous medium containing a small amount of an alkali metal carbonate or bicarbonate as an additive incapable of adversely affecting the polymerizability of the product acrylamide, and also, under some conditions, an organic carboxylic acid (e.g., see U.S. Pat. No. 4,343,900, British Patent Specification No. 2,062,625, German OLS (laid open) No. 3,037,009, French Pat. No. 2,466,506); and (2) a method which allows the reaction to proceed in a salt-free aqueous medium while maintaining cellular enzymes in a stable state, which comprises using immobilized cells entrapped with a cationic acrylamide polymer gel (e.g., see U.S. Pat. No. 4,421,855, British Patent Specification No. 2,086,376, German OLS (laid open) No. 3,132,493, French Patent Application (laid open) No. 2,488,908 ).
However, the salt concentration in the former method (1) is about 0.1 wt. %, as seen in the examples of the references in the former (1). This concentration was selected while giving priority to the matter of the swelling of immobilized cells in columns for continuous reaction, and therefore it is still a relatively high salt concentration, although the salt selected does not particularly adversely affect the quality of the product, i.e., the aqueous acrylamide solution. The combined use of an organic acid such as acrylic acid is also undesirable when high-purity aqueous acrylamide solutions are required, since the addition of such acid constitutes the addition of a byproduct of the reaction. Therefore, if aqueous acrylamide solutions of higher quality (purity) are desired, it is necessary to search for a compound effective even in lower concentrations.
The latter method (2) was proposed as a method allowing the reaction to proceed in the substantial absence of any salt while maintaining cellular enzymes in a stable state. In method (2), the cellular enzyme has improved stability in the salt-free reaction system as compared, for instance, with previously known polyacrylamide-immobilized cells. However, it is undeniable that the cellular enzyme stability to endure the reaction over a prolonged period of time in method (2) is somewhat inferior as compared with phosphate buffer reaction systems.